Role of Endogenous Nitric Oxide in Airflow Obstruction in Smokers (Materials and Methods)

12Dec 2013

The experiments were approved by the human ethics committee of our university, and informed consent for participation was obtained from each subject. Nineteen healthy volunteers (all male) were allocated to two groups (Table 1): nonsmoking (n=9, mean [SE] 32.3 [3.4] years) and smoking (n=10,35.1 [1.7] years) subjects. Subjects with an asthmatic component, hypertension, or respiratory infection within the 2 weeks preceding the study were excluded. Smokers were asked not to smoke during the 2-h period preceding NO measurements. Spirometry was performed just before NO measurements using an autospirometer according to the standards established by the American Thoracic Society. The subjects were asked to breath through a mouthpiece, and a nose clamp was used. A T-valve was connected to the mouthpiece to allow breathing of synthetic NO-free air (<1 ppb; Takachiho Chemicals; Kyoto, Japan), and all subjects inhaled this NO-free air throughout the experiments.

NO in exhaled air was continuously analyzed in samples taken from the mouthpiece for single-breath measurement of mixed exhaled air. After flushing the analyzer with NO-free compressed air, all subjects were asked to breath through the mouthpiece for 2 to 3 min; they were then asked to hold their breath for 0 s, 5 s, 10 s, and 15 s, sequentially, starting at the end of normal inspiration, and then to exhale slowly. This procedure was repeated 3 times, allowing 1 min of normal breathing between each repetition.

Exhaled NO was measured on a chemiluminescence analyzer (CLM- 500; Shimazu; Kyoto, Japan) sensitive to NO from 2 to 1,000 ppb, adapted for online recording of NO concentration. A sample gas flow is 500 mL/min in this chemiluminescence system. The feature of this system obviates the need to collect air in a reservoir; therefore, it is very sensitive and its reproducibility is better. Results were displayed on a chart recorder and compared with the signal generated from a calibration mixture of 443 ppb NO in nitrogen diluted by precision flowmeters. Values are expressed as mean±SE. Differences between two groups were compared using one-way analysis of variance followed by the Fisher Test. The correlation coefficient was calculated using the least-squares method. Statistical significance was set at p<0.05.